Wednesday, March 18, 2009

Analgesic Action of a Sustained Release

Using hard capsules (SR318B) developed as a sustained release diclofenac sodium (DF-Na) preparation foronce-daily administration, we investigated the persistence of the analgesic effect after oral administration in thecanine urate-induced gonarthritis model. In the control group, injection of 2% urate into the knee joint induced gaitdisorder due to pain 2 hr after administration and thereafter. Gait disorder peaked 6 hr after urate injection, andgradually recovered after 12 hr. In the treatment group, SR318B at 1.0 mg DF-Na/kg body weight was orally administered6 hr before urate injection, and the walking score significantly decreased 2 hr after urate injection comparedwith the control group (p < 0.05). Although the analgesic effect was not observed at the peak of urate-induced pain,the walking score significantly decreased 14, 16, and 18 hr after urate injection compared with the control group(p < 0.05). The plasma diclofenac (DF) concentration peaked 6 hr after SR318B administration, and decreased toabout 1/3–1/5 12–18 hr after administration (peak of urate-induced pain), and the plasma level was below the quantificationlimit in three of five animals 24 hr after administration. DF was detected in the synovial fluid 24 hr afteradministration in all animals and the concentration was 0.03 ± 0.01 μg/ml (mean ± standard error). The abovefindings showed that the SR318B exhibits a persistent analgesic effect in a canine urate-induced gonarthritis model.Not only DF in the plasma but also the DF that transferred to the synovial fluid may be involved in this persistentanalgesic effect.Key words —–— diclofenac sodium, urate-induced gonarthritis, sustained release preparation, dogINTRODUCTIONDiclofenac sodium (DF-Na) is a phenylacetatenon-steroidal anti-inflammatory agent synthesizedby Ciba-Geigy of Switzerland in 1965 (Fig. 1). InJapan, sustained release capsules for twice-daily administrationhave been developed by SS PharmaceuticalCo., Ltd. (Japan) and used to treat rheumatoidarthritis, osteoarthritis, and lumbago.In order to reduce adverse effects, persistent drugeffect, and improve patient compliance, our aim wasto develop a preparation for once-daily administrationby inhibiting a rapid increase in the blood DFNaconcentration and maintaining the blood levelfor a prolonged time. We designed four preparationmethods: 1) hard capsules containing rapid releasegranules and granules coated with a combinationof enteric and water-insoluble polymers; 2) hardcapsules containing rapid release granules and entericfilm-coated plain granules combined with anorganic acid; 3) hard capsules containing granulescoated with a combination of water-insoluble andwater-soluble polymers; and 4) hard capsules containinggranules double-coated with water-insolubleand enteric films. We prepared 3–4 prototypes usingeach method and performed pharmacokineticstudies using beagles, and selected one preparationfrom those prepared from each method.1) Next, weadministered the four preparations to healthy adultmen and investigated the safety and pharmacokinetics.The hard capsules containing rapid release granulesand enteric film-coated plain granules combinedwith an organic acid at a ratio of DF-Na of 3 : 7 werethe most appropriate for the sustained release preparationfor once-daily administration based on thepharmacokinetics of diclofenac (DF).Thus, we orally administered this sustained re-*To whom correspondence should be addressed: Department ofHygienic Chemistry, College of Pharmacy, Nihon University,7–7–1 Narashinodai, Funabashi, Chiba 274–8555, Japan. Tel.:+81-47-465-5694; Fax: +81-47-465-5637; E-mail: tezukam@pha.nihon-u.ac.jpNo. 5 465lease preparation containing diclofenac (SR318B)to a canine urate-induced gonarthritis model2) andinvestigated the persistence of the analgesic effect.MATERIALS AND METHODSMaterials —–— The test substance SR318B contains89.98 mg of rapid release granules and52.68 mg of sustained release granules per capsule,and 7.5 mg and 17.5 mg of DF-Na were containedin the rapid release and sustained release granules,respectively. The capsules were stored at room temperatureuntil use.Urate crystals were provided by SSP Co., Ltd(Japan). Heparin sodium solution for injection(Shimizu Pharmaceutical Co., Ltd., Japan), pentobarbitalsodium (Tokyo Kasei Kogyo Co., Ltd., Japan),water for injection, and physiological saline(Otsuka Pharmaceutical Factory, Inc., Japan) wereused.Animals —–— Ten male beagles aged six monthswere purchased from Nalc Co. (body weight: 8.57–10.03 kg). The animals were individually housedin metal bracket cages under conditions at 21–26°Ctemperature, 44–66% humidity, and 12-hr lighting(8:00–20:00). Animals were given about 300 g ofsolid food (LABO D STOCK, Nihon Nosan Kogyo,Co., Japan) per day and tap water ad libitum, andquarantined and acclimatized for 10 days. Animalsthat showed no abnormalities during the quarantine/acclimation period were used.Methods —–—Establishment of dosage and timing of urate administration:In the preliminary study in which 0.3and 1.0 mg DF-Na/kg body weight were orally administeredto dogs, the analgesic effect was observedat a dose of 1.0 mg/kg, and a small amount of DFwas detected in the plasma and synovial fluid 24 hrafter administration. Therefore, 1.0 mg/kg was selectedfor the dose. Since 2% urate crystal suspensionin physiological saline caused severe persistentgait disorder, we considered that to evaluate theanalgesic effect of SR318B, it is necessary to injecturate into the joint when the plasma DF concentrationis high, and selected 6 hr after administrationof the test substance for the time of administeringurate.Test substance administration and judgment ofanalgesic effect: Using five dogs per group, SR318Bwas orally administered at a dose of 1.0 mg/kg bodyweight between 21:00 and 22:00, and about 30 mlof water was immediately administered orally. Theamounts of the rapid release and sustained releasegranules contained in the capsule were adjusted bybody weight for each dog. 6 hr after administrationof SR318B, 0.5 ml of 2% urate crystal suspensionin physiological saline was injected into the kneejoint to induce inflammation, and walking was observed2, 6, 12, 14, 16, and 18 hr after injection ofurate. Walking was evaluated by five steps: normalwalking (0 point), mild claudication (1), moderateclaudication (2), walking on tiptoe (3), and walkingon three legs (4), and the mean score was obtainedin the control and SR318B treatment groups.Measurement of plasma and synovial diclofenacconcentrations: About 2 ml of blood was collectedfrom the cephalic vein using a heparinized syringe2, 6, 12, 18, and 24 hr after administration in fiveanimals in the SR318B treatment group. At the finalobservation, synovial fluid was collected fromthe knee joint in all animals.The collected blood and synovial fluid were centrifugedat 4°C, 3000 rpm for 10 min, and the obtainedplasma and supernatant of synovial fluid werestored at –20°C until analysis.The plasma and synovial DF concentrations weremeasured by high performance liquid chromatography.1)Statistical analysis: The mean and standard errorof the measured values were calculated in eachgroup. For analysis of significance in comparisonof the walking score between the SR318B treatmentand control groups, Wilcoxon rank sum test wasused.3) A difference with a significance level lessthan 5% was regarded as significant.